An affinity separation can be defined as any separation achieved by employing the specific binding of one molecule by another. Bioaffinity separation is defined as an affinity separation in which one of the components involved in the affinity reaction is biologically active or is of biological interest. Bioaffinity separations generally involve at least one biomacromolecule, such as a protein or nucleic acid, as one of the components of the binding pair. Examples of such bioaffinity binding pairs include: antigen-antibody, substrate-enzyme, effector-enzyme, inhibitor-enzyme, complementary nucleic acid strands, binding protein-vitamin, binding protein-nucleic acid; reactive dye-protein, reactive dye-nucleic acid; and others; the terms ligand and binder will be used to represent the two components in specific bioaffinity binding pairs.
Affinity separations are generally considered to require the use of solid carriers derivatized with a ligand or binder. These separations can be carried out as batch processes or chromatographic processes with the latter generally being preferred. Affinity chromatography is well known and has been reviewed, for example, in C. R. Lowe, "An Introduction to Affinity Chromatography", North Holland publishing Company, Amsterdam, N.Y. 1978. Lowe describes the characteristics desirable in a solid support to be used in an affinity separation. According to Lowe, the solid support should form a loose, porous network to allow uniform and unimpaired entry and exit of large molecules and to provide a large surface area for immobilization of the ligand; it should be chemically inert and physically and chemically stable; and the support must be capable of functionalization to allow subsequent stable coupling of the ligand. Additionally, the particles should be uniform, spherical and rigid to ensure good fluid flow characteristics.
The list of support materials suitable for affinity chromatography is extensive and will not be reviewed here (see Lowe, 1978, for a partial listing). It is not generally possible for a given support to achieve all of the above objectives. One requirement faced in preparing affinity supports from any carrier is the efficient and stable attachment of the ligand or binder to the carrier. The most common method employed is covalent attachment generally by modification of the carrier surface with a reactive reagent which then covalently bonds to the ligand or binder. Representative examples of this approach are given by Weetal [Methods in Enzymology, Volume XLIV: Immobilized Enzymes, Chapter 10, 134. Ed. K. Mosbach, Academic Press, New York, (1976)]. The major disadvantages of this approach are as follows: modification of the surface properties of the carrier which frequently results in increased nonspecific binding of unwanted proteins; inactivation of a significant portion of ligands or binders being bound; and the permanence of the attachment preventing recovery of scarce or expensive ligand or binder.
Another common attachment method is the nonspecific adsorption of the ligand or binder to the carrier. This approach is reviewed by Messing [Methods in Enzymology, Volume XLIV:Immobilized Enzymes, Chapter 11, 149, Ed. K. Mosbach, Academic press, New York, (1976)]. The major disadvantages of this approach are: relatively weak attachment, some or all of the bound ligand or binder is generally released during use; and partial inactivation of the ligand or binder being attached to the carrier. Despite these disadvantages this approach is still widely used due to its inherent simplicity.
Fluorocarbon polymers have been used as carriers to which ligands have been attached by adsorption [U.S. Pat. No. 3.843 443 issued to Fishman on Oct. 22, 1974; WO 8603-840-A filed by Rijskuniv Groningen; and Siergiej, Dissertation Abstracts, Int. B., Volume 44, 153 (1983)]. No attempt was made to modify the ligands to effect a specific interaction between the ligand and the carrier. Sakagani et al. [EP No. 0,011,504, published July 20, 1983] disclose the use of electrodeposition to attach ligands to fluoropolymer ion-exchange membranes. Again, no attempt was made to modify the ligand to effect a specific interaction between the ligand and the carrier.
Busby et al. (U.S. Pat. No. 4,317,879, issued Mar. 2, 1982) disclose the covalent attachment of an enzyme, glucose oxidase, to a fluorocarbon membrane through paraformaldehyde linking.
Hato et al. (U.S. Pat. No. 4,619,897, issued Oct. 23, 1986) disclose the immobilization of enzymes onto a fluorine resin membrane which is made hydrophilic on one side by the penetration of a perfluoroalkyl surface active agent to a prescribed depth. The asymmetrically functional membrane thus obtained is then treated with an enzyme and a crosslinking agent such as glutaraldehyde to achieve enzyme immobilization. The product could be utilized as an enzyme electrode.
Copending U.S. Pat. application, Ser. No. 863,607, filed May 15, 1986, discloses perfluorocarbon fluid-based liquid supports prepared by partitioning perfluoro-substituted ligands or binders to the surface of droplets of an emulsion of liquid perfluorocarbons.
Affinity separations often form a component part of other processes. One example is their use in heterogeneous immunoassays. Here the affinity separation is used to capture an analyte from a complex mixture such as serum or plasma. After capturing the analyte, the contaminants are washed away and the analyte is detected using well known assay protocols.
Some common solid supports in this area are plastic spheres (beads), interiors of plastic test tubes, interiors of microtitre plate wells, magnetic particles, and porous glass particles. One disadvantage of these systems is the difficult and inefficient attachment of ligand or binder to the support.
Certain separation problems have been traditionally dealt with by liquid-liquid extractions. For example, in nucleic acid hybridization assays, requiring purified nucleic acid, a nucleic acid from the sample, such as DNA or RNA, needs to be bound to a solid support. To obtain the nucleic acid to be probed it must first be released from a cell (if within a cell), by lysis, then extracted from the lysate. The most common extraction technique uses an aqueous phenol/chloroform mixture (Maniatis et al., Molecular Cloning: A Laboratory Manual, pp. 458-9. Cold Spring Harbor Laboratory, 1982). Proteins, which are the major component of the lysate, tend to interfere with the extraction. Following extraction of the nucleic acid, excess phenol must be extracted with ether and then the ether evaporated. The nucleic acid containing solution is then concentrated prior to deposition on a solid support; see, for example, Church et al, proc. Nat. Acad. Sci. USA, Volume 81, 1991 (1984). This is a tedious and hazardous process with many opportunities for material losses along the way.
Because affinity separation is a powerful technique and because currently available supports suffer from various disadvantages, there is a need for improved supports. These should have the following properties: physical and chemical stability; chemical inertness; compatibility with a variety of biological samples; utility in batch and chromatographic applications; high surface area; ability to allow high flow rates in chromatographic applications; ability to provide for ready and stable attachment of ligands or binders to the surface; and allow simple efficient regeneration of the support.